The invention relates to determining the concentration of analyte in a liquid test sample by immunochromatography techniques.
Immunochromatographic strip formats have become increasingly popular for qualitative, semi-quantitative and quantitative assays that use visual detection schemes. This type of immunoassay involves the application of a liquid test sample suspected of containing an analyte to be detected to an application zone of an immunochromatographic test strip. The strip includes a matrix material through which the liquid test medium and analyte suspended or dissolved therein can flow by capillarity from the application zone to a capture zone where a detectable signal, or the absence of such, reveals the presence of the analyte. Typically, the strip includes a means for immunospecifically binding the analyte to be detected with its specific binding partner that bears the detectable label. In one such scheme, as disclosed in U.S. Pat. No. 4,446,232, the strip contains an enzyme labeled, mobile binding partner for the analyte which is in a zone of the strip downstream from the sample application zone. If analyte is present in the test sample, it combines with its labeled binding partner to form a complex that flows along the strip to a detection zone containing a substrate for the enzyme label capable of providing a colored response in the presence of the enzyme label. The strip contains another zone in which analyte is immobilized, so that the labeled binding partner which does not combine with analyte, due to the absence of sufficient analyte in the sample, is captured and thereby inhibited from reaching the detection zone. There have been published various modifications of this technique, all of which involve competitive specific binding systems in which the presence or absence of analyte in the test sample is determined by the detection or lack thereof of labeled binding partner in the capture zone.
In European patent application EP 0 462 376 there is disclosed a procedure in which signal at the capture site and conjugate collection site of an immunochromatographic strip are detected and the analyte concentration is determined by the intensity of the signal at the capture site relative to the signal at the recovery site. Also of interest in this regard is U.S. Pat. Nos. 5,569,608, 6,183,972 and 6,436,721, each of which is incorporated herein by reference.
Most lateral flow immunochemical tests are also provided with a procedural control that typically consists of an anti-species antibody (to the antibody attached to the colored particle) bound to the membrane distal to the test line. Appearance of a colored line confirms that the correct procedures were used. Thus, it confirms that sample was added, it mixed with and solubilized the colored particle dried to the pad and the complex flowed through the membrane resulting in binding of the colored particle to the control line. The sample then continues to move up the strip to the control band that contains an immobile band of anti-species IgG to produce the control line.
Some manufacturers also provide a reference line that is located midway between the test and control lines. See, SureStep™ hCG Combo (II) Pregnancy Test, Product Insert, Catalog #6018, Applied Biotech, Inc., San Diego, Calif. The reference line is made by applying a fixed concentration of another nonspecific immunoglobin to the membrane midway between the test and control line. To the pad, which contains the human chorionic gonadotropin (hCG) antibody attached to a colored particle, is added another colored particle to which an antibody to the reference immunoglobin is attached. The amount of nonspecific immunoglobin and concentration of colored particle is adjusted to result in a reference line having an intensity equivalent to that of the test line's sensitivity claim for pregnancy detection. Thus, a test line's intensity equal to or greater than the reference line intensity would be indicative of pregnancy.
However, unlike serum samples, whose compositions are typically very consistent in terms of, for example, pH, protein concentration and ionic strength, urine samples are considerably more variable in composition. These differences represent interfering factors that can impact the immunochemical binding and influence the accuracy of the result. Many manufacturers formulate their tests to compensate for some of these sample differences, for example, pH and protein levels, by drying the colored particle in a buffer containing protein. However, additional interfering factors exist, for example, urine specific gravity (SG), which impact the accuracy of lateral flow immunochemical tests by interfering with binding of the analyte to its complementary binding reagent. Comparison of test line intensity to reference line intensity is of little value unless the immunochemical reagents for the reference line are identical to those for the test line and are impacted, to the same degree, by variations in interfering factors such as specific gravity.
Thus, there exists a need for a reference reagent system similar to that utilized for the test reagent system such that interferences in immunochemical binding show parallel effects on both systems. Ideally, the intensity of signal observed in the reference region intensity would parallel that of the test region so as to ensure greater fidelity of the result. The present invention satisfies this need and provides related advantages as well.